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Unraveling the Hook Effect: A Comprehensive Study of High

Antigen Concentration Effects in Sandwich Lateral Flow
Immunoassays
Georgina M. S. Ross, Daniel Filippini, Michel W. F. Nielen, and Gert IJ. Salentijn*
Cite This: Anal. Chem. 2020, 92, 15587−15595 Read Online

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ABSTRACT: Sandwich lateral flow immunoassays (LFIAs) are

limited at high antigen concentrations by the hook effect, leading
to a contradictory decrease in the test line (T) intensity and false-
Downloaded via 186.249.229.86 on September 6, 2022 at 17:15:47 (UTC).

negative results. The hook effect is mainly associated with the loss
of T, and research focuses on minimizing this effect. Nevertheless,
the control line (C) intensity is also affected at higher analyte
concentrations, undesirably influencing the T/C ratio in LFIA
readers. The main aim of this work is to identify and understand
these high antigen concentration effects in order to develop
ubiquitous strategies to interpret and mitigate such effects. Four
complementary experiments were performed: performance assess-
ment of three different allergen LFIAs (two for hazelnut, one for
peanut) over 0.075−3500 ppm, LFIAs with C only, surface
plasmon resonance (SPR) binding experiments on the immobilized control antibody, and smartphone video recording of LFIAs
during their development. As antigen concentrations increase, the C signal decreases before the T signal does, suggesting that
distinct mechanisms underlie these intensity reductions. Reduced binding at the C occurred even in the absence of T, so the upfront
T does not explain the loss of C. SPR confirmed that the C antibody favors binding with free labeled antibody compared with a
labeled antibody−analyte complex, indicating that in antigen excess, binding is reduced at C before T. Finally, a smartphone-based
video method was developed for dynamically monitoring the LFIA development in real time to distinguish between different
concentration-dependent effects. Digitally analyzing the data allows clear differentiation of highly positive samples and false-negative
samples and can indicate whether the LFIA is in the dynamic working range or at critically high concentrations. The aim of this work
is to identify and understand such high antigen concentration effects in order to develop ubiquitous strategies to interpret and
mitigate such effects.

ateral flow immunoassays (LFIAs) have revolutionized

L consumer diagnostics, translating laboratory-based immu-
noassays into affordable and accessible home testing devices.1
Sandwich-format LFIAs utilize two bivalent monoclonal
antibodies (mAbs) to capture and detect large multivalent
targets, such as allergens.2 In microplate-based immunoassays,
capture mAbs are directly immobilized onto the solid support
of the microwell;3 in LFIA, the capture mAb is shaped into a
test line (T). A labeled secondary mAb, which generally binds
to a different or repeating epitope on the antigen, forms a
sandwich complex with the antigen and the capture antibody.
The label yields a measurable, often optical signal. In a
sandwich LFIA’s working range, the T signal increases with an
increase in the target antigen concentration; the naked eye can
qualitatively read this. However, researchers have known since
19744 that an excess antigen concentration leads to saturation
of available binding sites on the bivalent capture and detector
mAbs, preventing the formation of a sandwich complex in the
T area, which in turn leads to a paradoxical loss of T signal
intensity.3,5−7 This disappearance of T is known as the hook
effect.8
In LFIA, in addition to the capture and detection mAbs, a
secondary species-specific antibody, capable of binding the
labeled detection mAb, is immobilized as a control line (C).9
The C informs the user that the test is valid, yielding a signal
regardless of the presence of an antigen. When analyzing LFIAs
with a digital optical reader such as a smartphone,10 the ever-
present C can be used to normalize the T, to correct for
experimental variables (T/C).11 The use of T/C thus assumes
that the C intensity is constant. However, it has been observed
that an increasing antigen concentration also leads to a
Received: September 3, 2020
Accepted: November 2, 2020
Published: November 13, 2020

© 2020 American Chemical Society https://dx.doi.org/10.1021/acs.analchem.0c03740

15587 Anal. Chem. 2020, 92, 15587−15595
Analytical Chemistry
decrease in the C intensity, while the T intensity still
increases.12−14 The loss of C compromises the reliability of
the T/C at high concentrations and yet remains to be fully
understood. In the literature, various concentration-dependent
effects are described under the hook-effect definition. See the
Supporting Information, Table S1, for a review of the
definitions given for the hook effect and the observed effects
in the literature (1974−2020). Despite the qualifying
characteristic of the hook-effect being a false negative result
(i.e., the absence of T), the definition is frequently also used to
describe effects causing the loss of the C.
There are numerous mitigation strategies to cope with high-
concentration effects in sandwich immunoassays:
The most apparent method is testing the sample, both
undiluted and diluted.15 If the diluted sample gives a stronger
T response than the undiluted sample, the undiluted result can
be considered as “hooked”.16 Dilutions allow adjustment of the
dynamic working range of an LFIA but also require additional
sample preparation, time, and material costs.17 Conversely,
changing the physical layout of the assay can prevent high-
concentration effects,2,18 essentially allowing for decoupled
reagent delivery.19 Still, the separation of reagent flow is crucial
to prevent premature mixing of the labeled antibody and
analyte.20 Alternatively, high-concentration effects can be
minimized by optimizing reagents, for example, by supple-
menting the immunoassay with one or more additional target
lines.6,21,22 Differentiation between artificially low (“hooked”)
and truly low concentration samples is possible by the real-
time monitoring of T and C development14 and can allow for
LFIA dynamic ranges to be expanded by orders of
magnitude.23
In this work, the aim was to first unravel the hook effect by
comprehensively elucidating how extreme antigen concen-
trations influence the LFIA test line and control line
development over time in three different allergen LFIAs. The
identification and understanding of how high-antigen concen-
trations influence LFIA signal development is crucial for any
developed sandwich LFIAs and will lead to ways to mitigate
such effects, such as the simplified dynamic smartphone-based
method presented here, ultimately leading to more reliable
testing.
■ EXPERIMENTAL SECTION
Three allergen LFIAs were developed for detecting peanut
(PA) and hazelnut (HA1 + HA2). Each assay has a different
sandwich pair of mAbs for their capture (T) and detector
carbon nanoparticle-labeled-antibody (CNP-mAb), selected
for their differences in sensitivity as observed in prior
works.24−26 All assays used goat anti-mouse (GAMaB) IgG
in PBS (pH 7.6; 1.2 mg/mL; AffiniPure F(ab’)2 Fragment) at
the C line (Jackson Immunoresearch Laboratories Inc., Sanbio,
Uden; The Netherlands) and were developed on nitrocellulose
membranes (140 CN; Unisart, Sartorius, Gottingen, Germany)
overlaid with an absorbent pad (Whatman, GE Healthcare,
Eindhoven, The Netherlands) and secured with a plastic
backing (G and L, San Jose, CA, USA); see Supporting
Information Protocol S1 and S2 for full details on CNP-mAb
labeling and LFIA preparation details. For the surface plasmon
resonance (SPR) biosensor assay, an amine coupling kit, pH
scouting kit, HBS-EP buffer, and CM5 sensor chips were
purchased from GE Healthcare (Uppsala, Sweden); see
Supporting Information Protocol S3 for further details on
the immobilization procedure.
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Reference Material Preparation. Standardized certified
reference materials and standard solutions for food allergens
are not currently available, and therefore, the total protein
extracts used here required in-house preparation.27 The
procedure for the protein extraction for generating total
peanut protein (TPP) and total hazelnut protein (THP) has
been described previously.25,26 Fresh aliquots were defrosted
on the day of the experiment, and the protein content was
checked with the NanoDrop (ND 3300, Isogen Life Sciences,
De Meern; The Netherlands) before use.
LFIA Readout. A qualitative assessment of LFIAs was made
by reading the developed signal with the naked eye;
quantitative readings were performed by smartphone detec-
tion.28 A custom 3D-printed smartphone holder was used to
shield up to three LFIAs from ambient light during the optical
measurements (See Figures 1, S1 and S2). A smartphone
Figure 1. Photographs of the 3D-printed smartphone holder for
recording LFIA signal development under controlled lighting: (A)
open side view to show where LFIAs and microwells are inserted; (B)
closed side view with the LFIAs inserted and smartphone attached;
and (C) head-on view of LFIA signal development on the phone
screen.
(Google Pixel 2 XL, Google, Mountain View, CA; USA) was
used to record images and videos of developing LFIAs. The
smartphone was attached to the frame of the holder for
support during the dynamic measurements (locked exposure,
fixed focus, and controlled illumination) at 30 frames per
second (fps) using OpenCamera (v1.47.3). Adapter (v2.1.6)
converted the videos into images of 1 fps. ImageJ29 was used to
split the images into their RGB (red, green, and blue) color
channels. Blue channel intensity values for the T and C were
subtracted from a background reading; the resulting corrected
blue channel pixel intensity (cBCPI) increased with increasing
line intensity.
Influence of Antigen Concentration on LFIA Signal
Development. All three LFIAs were tested in a concentration
range spanning 5 orders of magnitude. The LFIAs were
inserted into microwells containing 99 μL of antigen in RB
(0.0075−3500 ppm of TPP or THP) and 1 μL of CNP-mAb.
Here, the endpoint images of T and C signals were used for
calculating T/C; LFIAs were left to develop for 40 min before
the results were recorded using a smartphone.
Influence of Antigen Concentration on Signal
Development in C-Only LFIAs. The LFIAs were tested
alongside an LFIA with only a GAMaB C, at concentrations
shown to diminish C intensity (5, 10, 25, and 50 ppm) (n = 2).
Additionally, the original LFIAs were run in a blank as a
negative control. The LFIAs were developed in a microwell
containing 99 μL of antigen in RB (50 ppm TPP, 5 or 50 ppm
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Figure 2. Extended calibration range of the three LFIAs, peanut assay [PA], hazelnut assay 1 [HA1], and hazelnut assay 2 [HA2] in increasing
concentration of the analyte (TPP or THP) spiked into RB (0.0075−3500 ppm). (A,D,G) Photographs after 40 min, (B,E,H) test and control
signal expressed in cBCPI, and (C,F,I) test line divided by the control line (T/C ratio). Error bars represent the standard deviation (n = 3).

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Analytical Chemistry
THP) and 1 μL of CNP-mAb; the LFIAs were developed for
30 min in a 3D-printed smartphone holder (Figure 1).
Sequential and Premixed Antigen-Binding Studied
by SPR. Biosensor chip immobilization was performed per the
manufacturer’s instructions; see Supporting Information
Protocol S3 for further details. Using SPR, the influence of
increased analyte concentration on the binding characteristics
of free CNP-mAb (and subsequently introduced analyte) or
premixed immunocomplex (CNP-mAb-analyte) toward im-
mobilized GAMaB was evaluated (n = 3). For sequential
measurements, 10 μL of CNP-mAb (diluted 1:99 in HBS-EP)
was injected across the GAMaB surface (flow rate of 25 μL/
min). After binding, 10 μL of analyte solution was injected. For
premix experiments, 10 μL of CNP-mAb and analyte solution
was injected at 25 μL/min. These experiments were performed
at concentrations of 0.1−3000 ppm. For regenerating the
surface, the flow was adjusted (50 μL/min) and 5 μL of 25
mM NaOH was injected, returning the signal to baseline.
Dynamic Monitoring of LFIA Signal Development.
The LFIAs were placed in microwells containing 99 μL of
antigen in RB (0.0075−3500 ppm of TPP or THP) and 1 μL
of CNP-mAb, inserted into the smartphone holder, and were
dynamically recorded for 30 min. The T/C was acquired at set
time points (5−30 min) by selecting frames from the video.
This was done for different concentrations [(PA; 0.015−3000
ppm), (HA1: 0.015−3000 ppm), and (HA2: 0.015−2000
ppm)]. Alternatively, the videos were imported into custom
python scripts (Python 3.8) for automated data analysis. In an
early video frame, regions of interest (ROI) were positioned
over the T and C, and as a background reading. Data
evaluation consisted of averaging the blue pixel intensity in the
ROIs across the entire video duration at 1 second data points.
The generated responses were exported in a comma-separated
value (.csv) format for easy importing into spreadsheet
programs. A second complementary python script corrected
the time response to assess LFIA signal development.
■ RESULTS AND DISCUSSION
Influence of Antigen Concentration on LFIA Signal
Development. As most reported LFIAs are tested within a
limited range and read after up to 20 min, high concentration
effects are not well documented, but it is known that excess
antigen concentrations can influence the signal development
time. Here, three different LFIAs were tested in a
concentration range spanning 0.0075−3500 ppm (Figure 2)
and developed over 40 min before their endpoint image was
recorded using a smartphone.
In all three assays, the T follows the same pattern across the
concentration range, as can be observed visually (Figure
2A,D,G) and numerically (Figure 2B,E,H). The T signal
depends on the capture of antigen followed by binding of
CNP-mAb, or on the binding of already complexed [CNP-
mAb-analyte] to the immobilized antibody. At 0 ppm, there is
no analyte present; therefore, no T signal develops. As the
analyte concentration increases [up to 100 ppm (PA), 10 ppm
(HA1), and 10 ppm (HA2)], the T intensity increases by
capturing more analyte and correspondingly more CNP-mAb
or by capturing larger, higher-order immunocomplexes. These
complexes form when multivalent antigens, such as hazelnuts
and peanuts, with numerous identical or distinct epitopes,30
bind several CNP-mAb particles, leading to the formation of an
intense T at high concentrations. Beyond these concentrations,
the T intensity instead starts decreasing, producing false
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negatives. This hook effect is unsurprising because, at extreme
antigen-excess, T is rapidly saturated by an accumulation of
unlabeled antigen, while the remaining mobile, excess antigen
binds with CNP-mAb without being captured on T.2
Comparably, the C signal arises because of the binding of
CNP-mAb. Therefore, in a blank sample (0 ppm), a clear C is
seen. At low concentrations, C captures free CNP-mAbs,
yielding an intense signal, as observed visually (Figure 2A,D,G)
and numerically (Figure 2B,E,H). In this range, C intensity
increases slightly with increasing antigen concentration,
possibly because of the binding of multiple CNP-mAbs to
the same multivalent allergenic protein, resulting in increased
signal intensity.31 At higher concentrations [roughly above 10
ppm (PA), 0.5 ppm (HA1), and 1 ppm (HA2)], the C
intensity decreases, while the T still becomes more intense.
This is reflected in the endpoint T/C metric (Figure 2C,F,I).
T/C increases along with T and rises further even at
concentrations causing C to decrease. This increase in T/C
widens the LFIA’s linear dynamic working range even when C
is affected by the concentration. Only after the hook-effect has
occurred, leading to a decrease in T, does the T/C drastically
drop. This trend is consistent across all three assays, despite
them detecting distinct antigens and using different mAb
sandwich pairs with diverse sensitivities and kinetics;24,25 as
well as from the body of literature describing high
concentration effects in LFIA (see Supporting Information
Table S12,12,14). A decrease in C must be caused by a
reduction in CNP-mAb binding, which could potentially be
due to preventing the arrival of CNP-mAbs at the C by the T
(investigated in the C-only section below) or decreasing the
avidity of the CNP-mAbs to bind at those sites, which is
assessed by the SPR experiments.32
Interestingly, as the concentration further increases [above
100 ppm (PA) and 250 ppm (HA1 and HA2)], the C intensity
partially reappears. At extreme concentrations, multivalent
proteins have a propensity to aggregate, potentially masking
their epitopes.33 Moreover, these antigen concentrations
probably hinder higher-order immunocomplex formation
because there is insufficient CNP-mAb available for binding
with the abundant antigen in larger complexes.12,34
Influence of Antigen Concentration on Signal
Development in C-Only LFIAs. In the extended calibration
range, specific concentrations caused the appearance of an
intense T and a comparably diminished C, affecting the T/C,
as was observed in Figure 2C,F,I. Possibly, at these moderately
high concentrations, the antigen, which is bound in the
immunocomplex with CNP-mAb, binds mostly at the T, thus
limiting the amount of CNP-mAb that can reach the C and can
interact with GAMaB.
Figure 3A−C shows the three assays tested at concentrations
observed to affect C development; Figure 3D shows the
cBCPIs (n = 2) of the C from the three variations of the assay,
for all the three assays. Both in the presence and absence of a
T, the C never reaches the full intensity it would reach in a
blank. Interestingly, the signal for the C in the regular LFIA
[green (PA), blue (HA1), and orange (HA2)] and the C-only
LFIA [checked green (PA), checked blue (HA1), and checked
orange (HA2)] are of similar intensity. However, 3D shows
that the intensity of C in the C + T LFIAs is often less than in
the C-only LFIAs, indicating that some binding of [CNP-mAb-
analyte] at T could have a minor contribution to the reduction
of binding at C. However, there is a more substantial C
intensity difference between the blank and antigen solutions
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Figure 3. Control line-only LFIAs. Control signal development in

LFIAs with a control line and a test line (C + T) and LFIAs with only
a control line (C). (A) Peanut assay (PA) C + T signal development
in a blank (B), C + T and C in 50 ppm TPP. (B) Hazelnut assay 1
(HA1) C + T and C signal development in B and in 5 ppm THP, (C)
hazelnut assay 2 (HA2) C + T and C signal development in B and in
50 ppm THP, and (D) signal intensity in B, C + T and C across all
three LFIAs as a cBCPI.

for each assay, at all tested concentrations. This emphasizes

that a reduction of binding at C must be due to the increased
antigen concentrations, causing the CNP-mAb to have a
decreased avidity for C.
Sequential and Premixed Antigen-Binding Studied
by SPR. Here, using SPR, we set out to elucidate whether
increased antigen concentration hinders the labeled [CNP-
mAb-analyte] immunocomplex’ ability to bind with the
GAMaB (premix) compared with whether increased antigen
concentration affects the binding of CNP-mAb decoupled
from the analyte. SPR typically is label-free,24 but antibody
labeling can alter essential binding characteristics.35,36 In SPR,
response units (RUs) are generated by the total amount of
material captured at the surface, compared with LFIA, where
the signal is made up solely from the binding of CNP-mAb to
C. Moreover, it is also important to note that these assays take
place on a very different time scale (i.e., LFIA; 40 min vs SPR;
40 s).
The RU’s reproducibly increased in all three assays following
the injection of CNP-mAb, as can be seen in Figure 4A−C, Figure 4. SPR responses showing binding to goat anti-mouse
where the black bar represents the free CNP-mAb binding with antibody of (A) total peanut protein [PA], (B) total hazelnut protein
GAMaB. Adjusting the concentration of the injected analyte in 1 [HA1], and (C) total hazelnut protein 2 [HA2] tested by
the second step again leads to a reproducible RU increase in all sequentially injecting the carbon nanoparticle-labeled antibody
assays, as can be seen by the increasing colored bar [green (CNP-mAb; black) followed by antigen [green (PA), blue (HA1),
(PA), blue (HA1), orange (HA2)]. Because these multivalent and orange (HA2)] compared against premixed CNP-mAb + antigen
antigens bind to the captured CNP-mAb, thereby increasing [checked green (PA), checked blue (HA1), and checked orange
the total mass of material bound to the chip surface, this (HA2])] (n = 3). Standard deviation is expressed as error bars (n =
increase in RU is unsurprising. 3).
Contrastingly, when simultaneously injecting CNP-mAb +
antigen (premix), the assays behaved differently [checked
green (PA), checked blue (HA1), and checked orange (HA2)] complex.2 This explains why high antigen concentrations cause
(see Supporting Information Figure S3 for an example of a reduction in binding toward GAMaB in both LFIA and SPR.
sequential and premixed TPP sensorgrams). Crucially, the The C signal is consistently reduced at a lower concentration
signal intensity in the premix approach is consistently lower than the T signal in LFIA. As soon as an assay is in antigen
versus the sequential approach at all tested antigen excess, a prerequisite to enter the hook-range, higher-order
concentrations. In excess analyte concentrations, [CNP-mAb- immunocomplexes would already have formed in the solution.
analyte] quickly forms, depleting the free CNP-mAb, which Formation of such complexes would deplete the amount of
would otherwise interact with C with higher avidity than the free CNP-mAb available for binding with C.
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Anal. Chem. 2020, 92, 15587−15595
Analytical Chemistry pubs.acs.org/ac Article

Figure 5. Dynamic smartphone monitoring of signal development for total peanut protein (PA), total hazelnut protein 1 (HA1), and total hazelnut
protein 2 (HA2) LFIAs. Control line signal development (A) [PA], (B) [HA1], and (C) [HA2]; test line signal development (D) [PA], (E)
[HA1], and (F) [HA2]; and T/C ratio development (G) [PA], (H) [HA1], and (I) [HA2] over 30 min at different concentrations [0.015−5 ppm
(PA)] [0.015−0.125 ppm (HA1 and 2)].

Therefore, for the hook effect at T to occur, the
concentration effect on the C must have already taken place.
The response in both [HA] assays is different from the [PA]
assay, although this is not surprising, considering that all the
assays use different antibodies with varied sensitivities and
detect distinct analytes. In the [PA], the total binding
decreases as antigen concentration increases, which is
consistent with the formation of a higher number of
immunocomplexes with reduced avidity to GAMaB. Pre-
viously, using SPR, Liang2 observed a concentration-depend-
ent decrease in labeled immunocomplex binding toward T, but
did not study how concentration influences complex binding at
C. Here, it is clear that there is a decrease in binding at
GAMaB with increasing TPP concentrations, seemingly
harmonious with a growing number of complexes. In both
[PA] formats, a decrease in C signal is observed at high
concentrations, but this stabilizes going up to 3000 ppm. In the
LFIA, one additional higher concentration (3500 ppm) was
also tested, and at this concentration, C partially recovered.
Contrastingly, in both [HA] LFIAs, we see a reappearance of
C at much lower concentrations than with [PA], which is
logical, considering that the [HA] LFIAs are more sensitive
than the [PA]. Likewise, both premixed HAs initially decrease
in RU with increasing concentration (<100 ppm), consistent
with the formation of higher-order complexes with reduced
avidity for C; these concentrations are also observed to cause a
15592
reduction in C in the corresponding LFIAs (Figures 2 and 3).
However, at higher concentrations, both HA assays instead
increase in RU; these concentrations likely cause protein
aggregation and have a relative scarcity of CNP-mAbs
compared to the overabundant antigen, inhibiting the
formation of higher-order complexes.37
Dynamic Monitoring of LFIA Signal Development.
The SPR data suggest that the binding of immunocomplexes
differs from the binding of free CNP-mAb, resulting in
variations of the C intensity across broad concentration ranges.
The usefulness of T/C for normalizing sandwich LFIA results
is impeded across this range when considering only the
endpoint analysis. However, recent research suggests that
additional information is available by monitoring the develop-
ment of the T/C over the entire assay duration.14 Figure 5
shows the signal development of the C (Figure 5A−C), T
(Figure 5D−F), and T/C (Figure 5G−I) of the LFIAs at a
range of TPP or THP concentrations [0.015−3000 ppm (PA)]
[0.015−3000 ppm (HA1) and 0.015−2000 ppm (HA2)] (see
Supporting Information Figure S4 for snapshots from the video
recording of developing LFIAs).
Within the assay working range [0.015−5 ppm (PA)]
[0.015−0.125 ppm (HA1 and 2)], the C always develops faster
than or at the same time as the T, resulting in a low, stable T/
C over time. With increasing concentrations [10−250 ppm
(PA)], [2.5−100 ppm (HA1)], and [2.5−400 ppm (HA2)],
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Table 1. Summary of Results of Concentration-Dependent Effects Studied in This Work

experiment
influence of concentration on
LFIA signal development
influence of concentration on
signal development in C only
LFIAs
sequential and
premixed antigen-binding
studied by SPR
dynamic monitoring of LFIA
signal development
purpose
investigate T and C and T/C development
(0.0075−3500 ppm)
determine how C signal develops without a T
determine the difference between premix and sequential
binding of CNP-mAb and antigen to GAMaB
(0.1−3000 ppm)
investigate T, C and T/C development over time at
varying concentrations
result conclusions
C increases at low [conc] hook effect is reproducible
T increases at high [conc], at extreme
[conc] T is lost (hook effect)
C decreases at a lower [conc] than T C decreases because of reduced
CNP-mAb binding or due to T
depleting CNP-mAb
C partially recovers at extreme [conc]
high [conc] prevents C from reaching high [conc] negatively affects C
the same intensity as in a blank
sequential [PA], [HA1], [HA2]: at high free CNP-mAb has higher avidity to
[conc] RUs increase GAMaB than [CNP-mAb-analyte]
complexes
premix [PA]: at high [conc] RUs
decrease
premix [HA1] + [HA2]: at low [conc]
RUs decrease and at high [conc] RUs
increase
dynamic working range: T and C at a dynamic monitoring of T and C can
similar time distinguish high concentration ef-
fect
high [conc]: T increases faster than C;
T/C increases and then decreases
extreme [conc]: no signal for approx.
10 min, then C increases

the speed of binding shifts, with the T developing more quickly
than the C. The T/C time development reflects this with a
sharp initial increase (5 min), when mostly T is present,
followed by a steady decline as the C belatedly develops and
the signal balances out. Outside of the dynamic working range,
when the T or C development is influenced by antigen
concentration, the final T/C becomes an unreliable metric.
Recently, Rey14 presented a method for monitoring LFIA
signal development, using a different label (i.e., gold nano-
particles), detecting another analyte (C-reactive protein), and
similarly observed an initial rise, followed by a decrease in T/C
over the assay duration at concentrations where the T develops
before the C. Interestingly, here we observed this trend across
all three LFIAs, albeit at different concentrations. Unfortu-
nately, Rey’s14 study primarily looked at T/C’s in a limited
antigen range (120−255 μg/mL), and as such only tested
concentrations that caused a delayed and diminished develop-
ment of C rather than the inhibition of T development. A
recent study23 consolidated Rey’s results by testing a different
antigen (hCG) and modeling and observing T/C development
at different antigen concentrations (0.5−500 IU/mL) over 10
min. They found that at low hCG concentrations, C initially
develops faster than T, with T/C increasing after C is saturated
and T keeps developing over 10 min. Conversely at high
concentrations T develops rapidly with C developing slowly,
causing T/C to intially rise and afterwards to steadily decrease
over the assay duration as T is saturated and C still increases.
which is in line with our own findings.
Additionally, we found that a delayed and weak C
eventually starts to develop after 10 min, giving a final low T/
C.
Critically, at these concentrations, the resulting low final T/
C overlaps with the T/C at much lower concentrations [0.015
ppm (PA), 0.015 and 0.03 ppm (HA1) and (HA2)];
misinterpretation of this could lead to reporting of a false
negative, as can be seen in Supporting Information Figure S6,
where the T and C signal development and resulting T/C are
compared for HA1 at 0.015 ppm and 2000 ppm. While the
final T/Cs at these concentrations are similar, a false negative
can be avoided by monitoring when the T and C develop; at
hook-effect concentrations, no signal develops on either line
for the first 10 min, causing a static T/C during this time.
Comparatively, at low concentrations in the dynamic working
range, the C signal develops rapidly and with a high intensity.
By using dynamic video acquisition, real and artificially low
concentration measurements can be differentiated, not only
based on signal intensity but additionally based on whether the
C or T develops first (Figure 5). Further, it is possible to
automatically generate these T and C development profiles
directly from the smartphone video (see Supporting
Information Figure S7 for automatic profiles for PA compared
with manual time-development graphs and Figure S8 for a
blank assay), using the python script. In addition to dynamic
intensity measurements, this allows for BCPI correction and
autobackground subtraction. Considering that the results
generated by the software correlate with the manually plotted
time responses, novice users can simply use the automated
results rather than carrying out the image analysis, corrections,
and background subtraction themselves.

development at higher concentrations also occurs when testing
LFIAs without a T [50 ppm (PA), 25 ppm (HA1), and 50
ppm (HA2)] (Supporting Information Figure S5), where the
C signal only starts developing after 10 min and then only with
a diminished intensity. As concluded from the SPR study,
reduction in C intensity is mainly caused by the formation of
(higher-order) immunocomplexes, which have reduced avidity
for C. While any T signal is weak, a diminished and delayed C
15593
■
CONCLUSIONS
The experiments, results, and conclusions drawn have been
summarized in Table 1; a schematic depiction of the
complementary experiments can be found in Supporting
Information Figure S9. To unravel the hook effect, we devised
an inexpensive, dynamic, smartphone-based method for
https://dx.doi.org/10.1021/acs.analchem.0c03740
Anal. Chem. 2020, 92, 15587−15595
Analytical Chemistry
directly identifying the concentration-dependent effects across
three different sandwich LFIAs. We comprehensively eluci-
dated how both the antigen concentration and time influence
signal development and allow us to differentiate between two
distinct concentration effects: (1) the reduced development of
a C in the presence of a rapidly and intensely developing T,
which occurs within the first few minutes and (2) the decrease
in signal development on either line for 10 min, followed by
the development of a C which increases in intensity for the
remaining assay duration. These trends were consistent across
all three assays.
Indeed, we discovered for the hook effect to occur on T, the
concentration effect at C must have already happened. Based
on our findings, we propose that a more appropriate definition
for the moderate-high concentrations, which lead to the loss of
the C in LFIA, would be the situation of free secondary antibody
depletion. In f ree secondary antibody depletion, higher-order
[CNP-mAb-analyte] immunocomplexes form, hindering the
signal development at C by reducing the CNP-mAb avidity for
binding. As the concentration of analyte increases further, the
assay starts to enter the hook-effect range. At these extreme
concentrations, the unlabeled analyte rapidly saturates T. We
have established that while the endpoint T/C is an appropriate
metric within the dynamic working range, outside of this range
when the test or control line is falsely diminished, the final T/
C is influenced. While a prediction algorithm is outside of the
scope of this paper, we appreciate that this would be a useful
advancement. Here, the automatically generated qualitative
binding profiles provide a simplified way for novice users to
monitor concentration effects without performing any image or
data analysis themselves.
Further, the current system can simultaneously analyze three
LFIAs, making it feasible to include an in-built quality control
LFIA; such a control would be highly relevant where non-
experts, such as allergic consumers, perform LFIAs. Ultimately,
the use of dynamic readout provides an inexpensive and direct
mechanism for identifying the high-concentration effects in
LFIA. The digital analysis of dynamic data allows clear
differentiation between the highly concentrated samples and
low concentration results. We foresee that this method should
have broad applicability for distinguishing false-negative results
in sandwich LFIAs.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.0c03740.
Overview of hook-effect definitions; protocols for CNP-
mAb labeling, LFIA preparation and SPR procedures;
3D-printed attachments, SPR sensorgrams, photos of
developing LFIAs, automatically generated kinetic
profiles, and a schematic overview of paper (PDF)
■
AUTHOR INFORMATION
Corresponding Author
Gert IJ. Salentijn − Wageningen Food Safety Research
(WFSR), Wageningen University & Research, Wageningen
6700 AE, The Netherlands; Laboratory of Organic
Chemistry, Wageningen University, Wageningen 6708 WE,
The Netherlands; orcid.org/0000-0002-2870-9084;
Email: gert.salentijn@wur.nl
15594
pubs.acs.org/ac Article
Authors
Georgina M. S. Ross − Wageningen Food Safety Research
(WFSR), Wageningen University & Research, Wageningen
6700 AE, The Netherlands; orcid.org/0000-0002-2145-
2386
Daniel Filippini − Optical Devices Laboratory, Division of
Sensor and Actuator Systems, IFMLinköping University,
Linköping S58183, Sweden
Michel W. F. Nielen − Wageningen Food Safety Research
(WFSR), Wageningen University & Research, Wageningen
6700 AE, The Netherlands; Laboratory of Organic
Chemistry, Wageningen University, Wageningen 6708 WE,
The Netherlands; orcid.org/0000-0003-4634-0249
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.0c03740
Author Contributions
G.M.S.R. conceptualization; methodology; validation; formal
analysis; investigation; data analysis; writing original draft;
writingreview and editing; project administration D.F.
software; review and editing; M.W.F.N. resources; review
and editing; supervision; funding acquisition; project admin-
istration G.IJ.S. conceptualization; methodology; writing
review and editing; supervision; project administration.
Funding
This project has received funding from the European Unionʼs
Horizon 2020 research and innovation program under the
Marie Sklodowska-Curie grant agreement no. 720325.
Notes
The authors declare no competing financial interest.
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